Project Summary: Macrophage regulation of tissue repair and fibrosis through sensing of extracellular matrix mechanics Tissue fibrosis is a pathologic accumulation of extracellular matrix (ECM) that impairs normal organ function. In the lung, fibrosis can develop following a wide range of insults, including infection, radiation, chemotherapy, and foreign bodies like asbestos, or it can be idiopathic, with no known inciting injury. Fibrosis in all organs has been estimated to account for one third of all natural deaths worldwide, yet its underlying pathophysiology remains poorly understood, and few effective therapies have been developed that target the fibrotic process. Fibrosis is a disease of persistent tissue repair: continued extracellular matrix deposition creates pathologic scar tissue. Macrophages are essential for all phases of the repair process, including bringing it to a close (called `resolution'). To regulate tissue repair and appropriately turn it off, macrophages are likely to monitor the state of the extracellular matrix. Preliminary studies have shown that, when macrophages are induced to participate in tissue repair, they are highly sensitive to the mechanical environment, as measured by gene expression. The data suggest that macrophages may sense the increasing stiffness of the extracellular matrix during the course of repair, in order to appropriately regulate it and avoid fibrosis. This proposal will test this model, interrogate the mechanism of ECM sensing, and determine the implications of this biology for the pathogenesis of fibrotic diseases, in particular idiopathic pulmonary fibrosis (IPF). The first aim of the project is to determine whether stiffness or another feature of the ECM is sensed by macrophages. To address this question, bone marrow-derived macrophages (BMDM) will be cultured within novel three- dimensional hydrogels (based on polyethylene glycol) in which ECM concentration and stiffness can be independently controlled. ECM-sensitivity of tissue-resident macrophages from the lung will also be compared. The second aim is to determine how macrophages sense ECM and how this leads to regulation of gene expression. This will be investigated by blocking candidate sensing pathways in vitro and ultimately in vivo and through transcriptomic and chromatin analyses. The third aim is to determine how macrophage sensitivity to ECM controls fibroblast behavior, which will be done through supernatant transfers and co-culture, and whether macrophages from IPF patients show aberrant sensing of or response to ECM. If successful, these studies will uncover a novel mechanism of extracellular matrix regulation, establish a clear role for macrophages in regulating tissue repair, and suggest targeted strategies for intervention in IPF.